Enzymatic process for preparing penicillins



United States Patent Ofice 3,152,050 Fatented 9st. 6, 1964 3,152,050 ENZYMATIC PROCESS FOR PREPARING PENIClLLlNS Norman H. Grant, Wynnewood, and Harvey E. Album,

West Chester, Pa., assigners to American Home Products Corporation, New York, N.Y., a corporation of Delaware No Drawing. Filed Dec. 27, 1962, Ser. No. 247,541 10 Ciaims. (Cl. 195-2S) This invention relates generally to an enzymatic process for preparing penicillins and new penicillins produced thereby, and more particularly, to a process for employing either of the enzymes ficin or papain in a method utilizing 6-aminopenicillanic acid for producing microbiologically active 6-(aminoacylamino)-penicillins.

It has previously been suggested that certain bacteria, e.g., E. coli may be utilized for linking the six-positioned amino group of 6-aminopenicillanic acid with carboxylic acids, e.g., o-chlorophenyl acetic acid, p-nitrophenylethyl acetic acid, and the like, or the sodium or potassium salts thereof, under pH conditions of from 4.0 to 5.5, with release of water. However, these known methods for synthesizing penicillins normally have the limitation that they are fairly specific for resynthesizing the same penicillins. I

that they will hydrolyze. Moreover, said methods usually involve the presence of a multiplicity of components in the reaction mixtures thus presenting problems with respect to isolation of the desired penicillin product. Accordingly, these methods have been of limited value in the preparation of known peuicillins and in the quest for producing new and useful penicillins.

We have now discovered a novel method for producing penicillins, which method also utilizes -aminopenicillanic acid, but affords a means for producing a much broader range of penicillins than has been possible with said known methods. Generally, our novel method for preparing penicillins comprise reacting 6-aminopenicillanic acid with a carboxylic donor of the group consisting of the amino acids and the amide, ester, peptide and nitrile derivatives thereof, in the presence of an enzyme of the group consisting of ficin, papain, and bromelain, at a pH of from about 4 to 8. The carboxylic donors of the group just described may for the most part be represented by the general formula:

wherein each of R and R" represents a member of the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, alkaryl-sulfonyl and acyl; R and R when joined complete a heterocyclic ring of the group consisting of pyrrolyl, imidazolyl, piperidinyl, piperazinyl, and substituted derivatives thereof, R" represents a member of the group consisting of hydrogen, lower alkyl and acyl, and Z represent a member of the group consisting of hydroxy, amino, alkoxy, aryloxy and cycloalkoxy.

Preferably, the reaction takes place in the presence of an activator for the enzyme employed. Activators particularly useful for such purpose are the alkali metal and alkaline earth metal salts of glutathione, thioglycolic acid, and boron hydride. It is further preferred that the reaction take place in an inert atmosphere such as nitrogen. In such cases, the inclusion of an enzyme activator in the reaction mass has not always been found necessary. 0n the other hand, when the reaction occurs in air where the SH group of the enzyme may be oxidized, use of an activator has been found necessary. To maintain the pH of the reaction mass Within a preferred range, it has been found advantageous to include a buffer such as sodium acetate-acetic acid and the like.

In proceeding with the method of the invention, it has generally been found most advantageous to dissolve the carboxyl donor, the -aminopenicillanic acid, the enzyme and the activator therefor (if included) in a sufiicient amount of buffer solution to assure a pH within the specified range. As is known in the art, a soluble salt of 6- aminopenicillanic acid, e.g., the sodium or potassium salt thereof, may be used in lieu of the acid in certain in stances. Thereafter, the system is preferably evacuated and the air replaced by the inert atmosphere. In this connection, use of carbon dioxide should be avoided since it tends to degrade the 6-aminopenicfllanic acid in the reaction mix. After initial mixing, the reaction is then preferably allowed to proceed under incubating conditions. Thus, the reaction may be permitted to occur at say 22 C. for say 18 hours without agitation. It has been found advantageous to reduce the pH of the reaction mass, subsequent to the incubation reaction, to a pH of 2.5, and then to extract the acidic mixture with an organic solvent such as ether, amyl acetate, or the like. The penicillin product may then be precipitated from the extracts by addition of a base such as potassium acetate in isopropyl alcohol, or extracted into water at about pH 6.0 to 8.0 and the product isolated by a suitable procedure, e.g., ion exchange, crystallization, lyophylization.

In preparing the reaction mixtures useful in the method of the invention, advantageously the carboxyl donor concentration may be in the range of 0.01-1.00 M, the 6- aminopenicillanic acid concentration may be in the range of 0.01-0.20 M, the pH may be in the range of 4.0-5.8, and the ionic strength of the solution (based on univalent bufier concentration) may be in the range of 001-10 The time of reaction may be from 15 minutes up to 48 hours at an incubation temperature of from 20-55 C. Under particularly advantageous conditions, the carboxyl donor concentration may be about 0.50 M, the 6- aminopenicillanic acid concentration 0.10 M, the pH from 4.8-5.0, ionic strength 0.12, the incubation temperature 37 C. The time of reaction may advantageously be 3 hours at donor concentration greater than 0.2 and 24 hours at donor concentration less than 0.2.

Of the carboxylic donors contemplated for use in the exercise of the method of the invention, there are noted such natural amino acids as alanine, arginine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, histidine, hydroxylysine hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, thyroxine, tryptophan, tyrosine and valine.

Examples of other amino acid compounds and derivatives suitable as carboxylic donors in the method of the invention are listed below:

E no CH2.THCO.NH

C 5H5C O .NH Benzoyl tyrosyl glycinamide Carnosine CH COgH CE2CONH CHsCHCONH NHg Alanyl glycyl glycine Hz (J 0 2H 0 H 0 0 NH O aHs- C O NH Benzoyl glycyl glycine CHaCHOOgH CBHFCHZO 0 ONE Carbobenzoxy alanine HNOHZCH3CH3CHCO2H HN=C 1 in,

zlll now-cradmcnoom Arginine glutamate Alanyl asparagine Other amino acids and ester, simple amide, and nitrile derivatives suitable as carboxylic donors for use in the method of the invention will occur to those skilled in the art. Similarly, other peptides than those listed are snitable as long as they, too, do not have a molecular weight in excess of 500.

The following examples. are illustrative of the invention and should not be considered as necessarily limitative thereof:

EXAMPLE 1 Twenty-four games of hippuric acid (benzoylglycine), 4.8 grams of 6-aminopenicillanic acid, 360 milligrams of sodium glutathione, and 600 milligrams of partially purified ficin were dissolved in 300 ml. of 0.1 M sodium acetate-acetic acid buffer of pH 5.0. The ficin used had been prepared by dissolving 100 g. Paul Lewis P/L 2011 ficin in 400 ml. of distilled water and dialyzing against 20 liters of distilled water with two water changes. The dialysis retentate was then freeze-dried and was ready for use in the described system.

The system was evacuated and the air replaced by nitrogen. After initial mixing, the reaction was allowed to proceed at 22 for 18 hours without shaking. 100 ml. of the reaction mixture, pH 5.1, was adjusted to pH 2.5 with 6 NHCl and filtered. The precipitate was stirred for 15 minutes with 100 ml. of ethyl ether, the ether layer was mixed with 30 m1. of 0.5 M potassium phosphate, pH 7.5, and the pH was adjusted to 7.0 by the addition of NaOH. The pH 2.5 filtrate was extracted twice with one volume of ether and the ether extracts were extracted with the phosphate buffer at pH 7.07.2. The aqueous solution, 35 ml., was adjusted to pH 2.5 and extracted twice with 10 ml. volumes of amyl acetate. The penicillin was precipitated by adding 3 ml. of 1 M potassium acetate in isopropyl alcohol to the amyl acetate extract. After washing with isopropyl alcohol, the product was dried.

ml. of the original reaction mixture was worked up as above, except that ether was replaced by n-amyl acetate throughout.

The two preparations were pooled, washed with isopropyl alcohol and dried, giving 295 mg. of benzamidoacetamidopenicillin.

Characterzzanon Property Expected Found B-lactam as 6-APA (Hydroxamate assay),

mg. mn- 0. 54 0. 54 0, percent 49. 3 47.1 H, percent 4. 3 4. 5 N, percent 10. 1 9.6 S, percent 7. 7 7. 5 M01. wt. (K assa 415 405 Units/mg. (Microbiological assay), K salt 198 [a] 25 +204. 5 LR 1 Benzoyl, fl-lactam, amide.

Balance Summary fl-lactam Penicillin Fraction as mg. units 6-APA Starting material 1, 600 960 Reaction mixture 1, 152, 000 Acid ppt., not ether extdn 150 55, 000 Acid filtrate, not ether ext 780 67, 500 Combined ether extracts 118 41, 000 Extd. by ether but not amyl acetate--. 25 9, 250 Penicillin product 56 21, 200 Starting material 2, 400 1, 440 Reaction mixture 1, 775 228, 000 Acid ppt., not amyl acetate ext 435 120, 000 Acid filtrate, not amyl acetate ext 975 19, 000 Acid ppt. extd. by amyl acetate.-- 57 18, 000 Acid filtrate extd. by amyl acetate 270 92, 000 Ext 1. but not re-extd. by amyl acetate 112 30, 000 Penicillin product 109 41, 600

EXAMPLE 2 A reaction mixture was prepared of components which in their final concentrations comprised ficin (1.6 mg./ ml.), glutathion (6.6 mg./ml.), N-carbobenzoxyglycyl-L- tryptophane (7.9 mg./ml.), 6-aminopenicillanic acid (4 mg./ml.), and potassium phosphate buffer (pH 7.0, 0.3 M). This system was shaken for 3.5 hours at 38. When assayed against Staph. aureus 209?, it showed 290 penicillin G units per ml. Controls, assayed fewer than 10 units/ml.

EXAMPLE 3 A reaction mixture system was prepared in which the components with their final concentraions, were ficin (1.6 mg./ml.), glutathione (6.6 mg./ml.), N-carbobenzoxy- DL-glutamine (5.6 mg./ml.), potassium -aminopenicillanic acid (5 mg./ml.), and sodium acetate bufier (pl-I 4.8, 0.1 M). This system was shaken for 18 hours at 38. Paper disk assays of 1/50 dilutions, using Staph. aureus 209? on a clear zone scale of 1+, 2+, 3+, and

V 4+, showed 3+ for the complete system.

EXAMPLE 4 A system was prepared identical to that of Example 3, except that the enzyme was papain instead of ficin. The results with respect to assay against Staph. aureus 209? were substantially the same as in Example 3..

3 EXAMPLE Two separate systems were prepared substantially respectively identical to those of Examples 3 and 4, except that in each case the carboxyl-contributing reactant was the enzyme activator, and sufficient butter to put the mixture at pH 4.85 .0.

Routinely, with the exceptions noted hereinafter, the mixtures were adjusted to pH 4.8 and incubation was con- N c bgbgnzgxy-DL no -leucine The aper disk assays, 5 tinued for 18 llOllIS at 38 C. Thereafter, aliquots 0f the performed as in Example 3, showed lysed zones measresultant compositions were diluted 1/25 and 1/50 as apd t 4+ i b th cases pears in the table below and then assayed against Staph. FXAMW ES aureus. The activity of -aminopenicillanic acid against L that organism was routinely assayed for comparison and An XLI1S1V$ series of reaction mixtures, generally slml- L 10 round to be insignificant. lar to those of the preceding examples, was prepared. 1n F U Thus, all these mixtures contained 6-aminopenicillanic Senes OI IeHCLIOH nllxtures, and specifically the caracid, an enzyme selected from the group consisting of y p p ln at data with respect to any ficin and papal-n a carboxylic donor of the group wnsist. variations in dilution, 6-am1nopenic11lan1c acid concening of the amino acids and ihe amide, ester and peptide 15 tration, pH and incubation time, together with the results derivatives thereof having a molecular weight no greater Of all Of The aforesaid y 31B given in tablfi below than 500 and sodium glutathione as enzyme activator, and wherein the numerical results refer to Oxford Units per sodium acetate-acetic acid buffer. In each case, the reacml, bracketed values indicate results with variations in tion mixture contained 5 mg./ml. of 6-aminopenicillanic dilution, NA means no lysed Zone, A means lysed zone, acid, 1 mg./ml. of the selected enzyme, a concentration AA means extensive lysed Zone, and AAA means excepof .03 M of the carboxylic donor selected, .3 nag/ml. of tionally extensive lysed zone.

TABLE Enzyme Used and Dilution v Variations in G-APA C0nc., Ex. N0. Garboxylic Donor Ficln Papam pH, and/0r Incubation Time Benzoyl araininwmifie 0.04M: 6-APA pH 4.64.7. Benzoyl glypinqmmp gg llvfisfifAPA pH 4.6-4]. Benzoyl arginine ethyl ester ggg' f 4 hours Benzoyl tyrosyl glycinamide (1/20:O.80). GnZPA pH 7.2, Carbobenzoxy glycyl phenylalanine (U OIOglJ la fi Benzoyl tyrosin mide pHT.0; 4 hours.

Benzoyl tyrosine ethyl ester }pH 7.0; 4 hours Carbobenzoxy glycinamide l 3).. pH 7.0; 4 hours. Carbobenzoxy glycyl glycinamide .4 pH 7.0; 4 hours. Carbobenzoxy glycyl leucine 83 q pH 7.0; 4 hours. Carbobenzoxy slycyl phenylalaninamide 1{)66JI 3S) }P hoursaroo nz y gl'i y trypt phan }D 7.0; 4 hours. Oarbobenzoxy phenylglycine 6 0 6 None. Phenylqlqnin mirlen D0. Leucine methyl ester Do. p-Toluenesullonyl erg-thine methyl ester D0. Acetyl tryptophan ethyl est r Do. Acetyl tyrosine ethyl ester Do. Alanyl aspar Do. Alanine ethyl ester Do. Acctyl phenylalminc D0. Acetyl proline Do. Histidine D0. Histirline methyl ester D0. 4,S-imidazoledicarboxylic acid D0. Homocysteine thiolactrme D0. Glycine anhydrlde Do. Phenylalanine methyl ester D0. fi-pheny ine D0. Sal-cosine anhydr D0. Lysine glutamate Dlo Guanidoacetic acid D0.

Q-Phenylacetoacetnnitrilp D0. Cphenylg1ycine A Do. Phenyla a.nine D0, N-(p-HydroxyphenyD-glycine D0. Leucyl tyrosine Do Tyrosine ethyl ester Do.

Carbobenzoxy serine D0. Oarbobenzoxy phenylanine. D0.

Benzoyl glycyl phenylalanine D0.

Tryptophan ethyl ester". D0. p-Nit-ro benzoyl glycylglycine Do.

Glycyl leucyl tyrosine D0. Oarbobenzoxy isoleucine D0. Carbobenzoxy glutamine Do. Carbobenzoxy norleucine D0. Glutamic acid dimethyl ester D0.

Acetyl phenylalme ethyl ester D0. Glycyl tyrmirmmide D0. Benzoyl glycine Do.

Benzoyl glycyl phenyl-alanine- Do. Arginine 2-pyrrolidone5-carboxylate D0.

Acetyl phenylalanyl tyrosine Do. Benzoyl 1rininp D Carnrvirw DO. Alanyl glycyl glycine D0. Benzoyl glycyl glycine D0. Carbobenzox-y alanine. D0. Arginine lutamate DQ- 7 ll EXAMPLE 66 Prepare 5 ml. of a reaction mixture which in its final concentration contains 1.6 mg./ml. of purified commercial bromelain, 5.2 mg./ml. benzoyl-DL-argininamide, 4 mg./ ml. of 6-arnino penicillanic acid, and 0.3 M potassium phosphate buffer, and has a final pH of 7.0. Shake this system for 3.5 hours at 38 C. Thereafter, conduct paper disc assays of dilutions, using Staph. aureus 209P on a cleared zone scale of NA, A, AA and AAA, to show AAA for the system and NA for the controls.

EXAMPLE 67 Prepare 5 ml. of another reaction mixture similar to that of Example 66, but replace the benzoyl-DL-argininamide with 3.3 mg./ml. benzoylglycinamide, and the phosphate bufi'er with sodium acetate buffer in final concentration of 0.1 M (and pH of 4.8). Conduct similar assays to obtain the same antimicrobial results to show that bromelain catalyses synthesis of benzamidoacetamido penicillin in this case.

As is now well known in the art, the penicillin compounds, as produced by the method of the invention, or in the form of their regular salts, or the acid-addition salts thereof, are of value as antibacterial agents and as therapeutic agents in poultry and mammals including man, in the treatment of infectious diseases caused by gram-positive and gram-negative bacteria upon either parenteral or oral administration, and as nutritional supplements in animal feed.

We claim:

1. A method for producing penicillins which comprises (A) preparing a reaction mixture containing (1) 6-aminopenicillanic acid, (2) a carboxyl donor of the group consisting of (a) the amino acids and derivatives thereof having the formula:

carbobenzoxy, carbobenzoxyaminoace' nitrocarbobenzoxy aminoacetoXy, benzoyl, benzoylglycyl, lower toxy,

alkylsulfonyl, lower alkarylsulfonyl, amino lower acyl, aminoacetylamino lower acyl, a-amido aryl lower acyl, a-- benzamido hydroxyaryl lower acyl; N-(a-amino lower alkyl) amino lower acyl; R' represents a member of the group consisting of hydrogen, lower alkyl,

and lower acyl;

R and R", when joined, complete a heterocyclic ring of the group consisting of pyrrole and carboxy pyrrole;

Z represents a member of the group consisting of hydroxy, amino, and lower alkoxy;

R and Z, when joined, forma thio lower alkyl chain; and

R and Z, when joined, complete a i2 sarcosine anhydride ring, at which time R is hydrogen and R" is lower alkyl; and (b) the nitrile and peptide derivatives of (a) having a molecular weight no greater than 500, and (3) an enzyme of the group consisting of ficin,

papain, and bromelain, at a pH from 4 to 8; and (B) incubating the reaction mixture for a period sufficient to bring about formation of a microbiologically active penicillin compound.

2. A method for producing penicillins as claimed in claim 1 wherein a bufier is included in the reaction mixture.

3. A method for producing penicillins as claimed in claim 1 wherein an activator for the enzyme is included in the reaction mixture.

4. A method for preparing penicillins as claimed in claim 1 wherein the incubating occurs under an inert atmosphere.

5. A method of producing penicillins which comprises (A) preparing a reaction mixture containing (1) 6-aminopenicillanic acid, (2) a carboxyl donor of the group consisting of (a) the amino acids and derivatives thereof having the formula:

wherein R represents a member of the group consist ng of hydrogen, lower alkyl, hydroxy lower alkyl, amino lower alkyl, amino lower acyl lower alkyl,

guanido lower alkyl, carboxy lower alkyl, lower alkoxy lower alkyl, aryl lower hydroxy-alkyl, B-indolyl 'lower alkyl, 4-imidazolyl lower alkyl, aryl, aryl lower alkyl, hydroxy-aryllower alkyl;

R" represents a member of the group consisting of hydrogen, lower alkyl, guanido lower alkyl, lower alkoxy, carbobenzoxy, carbobenzoxyaminoacetoXy, nitrocarbobenzoxyaminoacetoxy, benzoyl, benzoylglycyl, lower alkylsulfonyl, lower alkarylsulfonyl, amino lower acyl, aminoacetylaminolower acyl, a-amido aryl lower acyl, os-lJBIlZ- amido hydroxy-aryl lower acyl, N-(aamino lower alkyl) amino lower acyl;

R'" represents a member of the group consisting of hydrogen, lower alkyl, and lower acyl;

R and R", when joined, complete a heterocyclic ring of the group consisting of pyrrole and carboxy pyrrole;

Z represents a member of the group consisting of hydroxy, amino, and loW- er alkoxy;

R and Z, when joined, form a thio lower alkyl chain; and

R and Z, when joined, complete a sarcosine anhydride ring, at which time R is hydrogen and R" is lower alkyl; and

(b) the nitrile and peptide derivatives of (a) having a molecular weight no greater than 500,

claim wherein the isolating step (E) comprises precipitating the penicillin compound from the extract by addition of a base.

7. A method of producing penicillins as claimed in claim 5 wherein the isolating step (E) comprises extracting into aqueous solution.

8. A method of producing penicillins which comprises (A) preparin a reaction mixture containing (1) from 0.0l0.02 M of 6-aminopenicillanic acid, (2) from 0.0l1.00 M of a carboxyl donor of the group consisting of (a) the amino acids and derivatives thereof having the formula:

R represents a member of the group consisting of hydrogen, lower alkyl, hydroxy lower alkyl, amino lower alkyl, amino lower acyl lower alkyl, guanido lower alkyl, carboxy lower alkyl, lower alkoxy lower alkyl, aryl lower hydroxyalkyl, ,B-indolyl lower alkyl, 4-imidazolyl lower alkyl, aryl, aryl lower alkyl, hydroxy-aryl lower alkyl;

R" represents a member of the group consisting of hydrogen, lower alkyl, guanido lower alkyl, lower alkoxy, carbobenzoxy, carbobenzoxyaminoacetoxy, nitrocarbobenzoxyarninoacetoxy, benzoyl, benzoylglycyl, lower alkylsulfonyl, lower alkarylsulfonyl, amino lower acyl, aminoacetylamino lower acyl, a-amido aryl lower [acyl, ct-benzamido hydroxyl-aryl lower acyl, N-(ocamino lower alkyl) amino lower acyl;

R represents a member of the group consisting of hydrogen, lower alkyl, and lower acyl;

R and R", when joined, complete a heterocyclic ring of the group consisting of pyrrole and carboxy pyrrole;

Z represents a member of the group consisting of hydroxy, amino, and lower alkoxy;

R and Z, when joined, form a thio lower alkyl chain; and

R' and Z, when joined, complete a sarcosine anhydride ring, at which time R is hydrogen and R" is lower alkyl; and

(b) the nitrile and peptide derivatives of (a) having a molecular weight no greater than 500,

(3) sufiicient of an enzyme of the group consisting of ficin, papain and bromelain to cause transpeptidation between components (1) and (2), and

(4) a buffer in amount to render the ionic strength of the solution in the range of 0.0ll.0 based on univalent bufier concentration at a pH of from 4 to 5.8; and (B) incubating the reaction mixture for a period of from 15 minutes to 48 hours at a temperature of from 25-55 C. to bring about formation of a penicillin compound. 9. A method of producing penicillins which comprises (A) preparing a reaction mixture containing (1) about 0.10 M of o-aminopenicillanic acid, (2) about 0.50 M of a carboxyl donor of the group consisting of (a) the amino acids and derivatives thereof having the formula:

wherein R represents a member of the group consisting of hydrogen, lower alkyl, hydroxy lower alkyl, amino lower alkyl, amino lower acyl lower alkyl, guanido lower alkyl, carboxy lower alkyl, lower alkoxy lower alkyl, aryl lower hydroxy-alkyl, B-indolyl lower alkyl, 4- imidazolyl lower alkyl, aryl, aryl lower alkyl, hydroxy-aryl lower alkyl;

R" represents a member of the group consisting of hydrogen, lower alkyl, guanido lower alkyl, lower alkoxy, carbobenzoxy, carbobenzoxyaminoacetoxy, nitrocarbobenzoxyaminoacetoxy, benzoyl, benzoylglycyl, lower alkylsulfonyl, lower alkarylsulfonyl, amino lower acyl, aminoacetylamino lower acyl, m-amido aryl lower acyl, a-benzamido hydroxy-aryl lower acyl, N-(aamino lower alkyl) amino lower acyl;

R' represents a member of the group consisting of hydrogen, lower alkyl, and lower acyl;

R and R", when joined, complete a heterocyclic ring of the group consisting of pyrrole and carboxy pyrrole;

Z represents a member of the group consisting of hydroxy, amino, and lower alkoxy;

R and Z, when joined, form a thio lower alkyl chain; and

R' and Z, when joined, complete a sarcosine anhydride ring, at which time R is hydrogen and R" is lower mkyl; and

(b) the nitrile and peptide derivatives of (a) having a molecular weight no greater than 500,

(3) an enzyme of the group consisting of ficin, papain and bromelain, in amount suflicient to cause transpeptidation between components (1) and (2), and

(4) a butter in amount to render the ionic strength of the solution at 0.12 based on univalent buffer concentration, at a pH of from 4.8 to 5.0; and

(B) incubating the reaction mixture at 37 C. for a period suflicient to bring about formation of a penicillin compound.

10. A method of producing penicillins which comprises (A) preparing a reaction mixture containing (1) about 0.10 M of 6-aminopenicillanic acid, (2) about 0.50 M of a carboxyl donor of the group consisting of (a) the amino acids and derivatives thereof having the formula:

wherein R represents a member of the group consisting of hydrogen, lower alkyl, hydroxy lower alkyl, amino lower alkyl, amino lower acyl lower alkyl, guanido lower alkyl, carboxy lower alkyl, lower alkoxy lower alkyl, aryl lower hydroxy-alkyl, B-indolyl lower alkyl, 4- imidazolyl lower alkyl, aryl, aryl lower alkyl, hydroXy-aryl lower alkyl;

R" represents a member of the group consisting of hydrogen, lower alkyl, guanido lower alkyl, lower alkoxy, carbobenzoxy, carbobenzoxyaminoacetoxy, nitrocarbobenzoxyaminoacetoxy, benzoyl, benzoylglycyl, lower alkylsulfonyl, lower alkarylsulfonyl, amino lower acyl, aminoacetylamino lower acyl, a-arnido aryl lower acyl, u-benzamido hydroxyaryl lower acyl, N-(aamino lower alkyl) amino lower acyl;

R' represents a member of the group consisting of hydrogen, lower alkyl, and lower acyl;

R and R, when joined, complete a heterocyclic ring of the group consisting of pyrrole and carboxy pyrrole;

Z represents a member of the group consisting of hydroxy, amino, and lower alkoxy;

1 5 R and Z, when joined, form a thio lower alkyl chain; and R and Z, when joined, complete a sarcosine anhydride ring, at which time R is hydrogen and R" is lower alkyl; and (b) the nitrile and peptide derivatives of (a) having a molecular weight no greater than 500,

(3) an enzyme of the group consisting of ficin,

papain, and brornelain,

(4) a bufler in amount to render the ionic strength of the solution at 0.12 based on univalent butter concentration, and

(5) an enzyme activator of the group consisting of the salts of glutathione, thioglycolic acid, and boron hydride,

at a pH of from 4.8 to 5.0;

(B) incubating the reaction mixture at 37 C, for a period of from 3 to 24 hours sufficient to bring about formation of a penicillin compound;

(C) thereafter, reducing the pH of the incubated reaction mixture containing the penicillin compound to a pH of about 2.5;

(D) extracting the acidic mixture with an organic solvent; and

(E) precipitating the penicillin compound from the extract by addition of a base.

References Cited in the file of this patent UNITED STATES PATENTS 3,047,467 Doyle et al July 31, 1962 3,079,306 Ofre et al Feb. 26, 1963 3,079,307 Kauffmann et a1 Feb. 23, 1963 3,088,880 Huang May 7, 1963 OTHER REFERENCES Kaufmann et al.: Antimicrobial Agents Annual, 1960,

Plenum Press, Inc., New York, pages 1-5.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,152,050 October 6, 1964 Norman H. Grant et al,

It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 2, lines 54 to 57, the formula should appear as shown below instead of as in the patent:

NH.CNH2 O.NH

' NH 6H5 Column-,3, lines 29 to 31, the formula should appear as shown below i ns'tead'.of- -a s in the patent:

column 6",, lines 62 to 65, the formula should appear as shown below instead of as in the patent:

column 7, lines 7 to 10, the formula should appear as shown below instead of as in the patent:

CH CHCO H 1\ NH (IJONH CH CH NH same column 7, line 58, for "games" read grams column 13, lines 29 to 31, the formula should appear as shownbelow instead of as in the patent:

Signed and sealed this lSth'day of July 1965.

(SEAL) Attest:

ERNEST W. SWIDER EDWARD J. BRENNER Attesting Officer Commissioner of Patents 

1. A METHOD FOR PRODUCING PENICILLIN WHICH COMPRISES (A) PREPARING A REACTION MIXTURE CONTAINING (1) 6-AMINOPENICILLANIC ACID, (2) A CARBOXYL DONOR OF THE GROUP CONSISTING OF (A) THE AMINO ACIDS AND DERIVATIVES THEREOF HAVING THE FORMULA 